Recombinant herpesvirus of turkeys and use thereof

ABSTRACT

The present invention provides a recombinant herpesvirus of turkeys modified by the presence of cDNA encoding the F protein of Newcastle disease virus under the control of a promoter. The poultry vaccine consisting of the recombinant herpesvirus of turkeys of the present invention can induce in chickens protective immunity against Newcastle disease virus.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides a recombinant herpesvirus of turkeys(rHVT) comprising cDNA of the F protein (F gene) of Newcastle diseasevirus (NDV) under the control of a modified chicken beta-actin promoter.

2. Description of the Related Art

Newcastle disease is one of the most fearful contagious diseases in thepoultry industry. Newcastle disease presents itself in many formsranging from high mortality to an asymptomatic form. Strains areclassified as (1) velogenic (high-virulence), (2) mesogenic(moderate-virulence), (3) lentogenic (low-virulence), and (4)asymptomatic (Alexander, D. J. In Diseases of Poultry 1997). Chickensinfected with velognenic forms of NDV become gloomy and lethargic in afew days and the mortality rate is from dozens to more than fiftypercent. Surviving birds often develop neurological symptoms such aswryneck or gyrospasm. One of the reasons the disease is so fearsome isthat chickens are susceptible to NDV regardless of age and thoseinfected with velogenic NDV show fulminant symptoms at all ages. SinceNDV is highly transmissible, every chicken must be disposed of at theoutbreak of the disease. The hennery should thoroughly be disinfected toprevent further infection. Strains in the mesogenic pathotype(moderate-virulence) are characterized by death in young chickens.Strains in the lentogenic pathotype (low-virulence) are characterized bymild respiratory infections and many of these strains are used toprepare vaccines for use in young chickens. Asymptomatic enteric strainsare usually isolated from the gut of chickens showing no disease(Alexander, 1997). Newcastle disease affects both chickens and turkeys,however the clinical signs in turkeys are less severe than in chickens(Alexander, 1997).

At present, live and inactivated vaccines are available for theprevention of Newcastle disease. These vaccines are effective but notfree from defect. The inactivated vaccine must be inoculated intobreeder hens in lay, repetitively. The live vaccine is mainly for youngchickens. However, long lasting immunity is not guaranteed for youngchickens, which have high maternal antibody levels. Repeatedadministration of live vaccines is sometimes detrimental to the healthygrowth of young chickens due to vaccine reactions causing mildrespiratory disease. Thus, a new type of vaccine, which is efficacious,free from adverse effects, and does not require repeated administration,is desirable for the poultry industry.

To meet the industry's wishes, Sakaguchi et al. (J. Virol. 74:3217-3226,2000; Vaccine 16:472-479, 1998) developed a recombinant Marek's diseasevirus serotype 1, which had the NDV F gene in the US10 region of thevirus genome. The obtained recombinant virus induced lasting protectiveimmunity against Newcastle disease in SPF chickens as well as inmaternal antibody positive commercial birds.

Marek's disease is another serious problem for the poultry industry.Against this disease, herpesvirus of turkeys (HVT) (Witter R. L. et al.Am. J. Vet. Res. 1970, 31, 525-538) has been most widely used as a safevaccine.

Until now, there have been several reports about the HVT-based Newcastledisease-Marek's disease bivalent vaccine. For instance, Morgan et al.(Avian Dis. 37:1032-1040, 1993; Vaccine 11:349-358, 1993; Avian Dis.36:858-870, 1992) constructed recombinant HVTs having the NDV F gene andexamined the efficacy of these recombinants as a Newcastle diseasevaccine. Macmillan et al. (Vaccine 14:469-477, 1996) constructedrecombinant HVTs expressing HN and F proteins of NDV and tested theserecombinants for efficacy. In both cases, vaccinated SPF chickens wereprotected against NDV, but not satisfactorily commercial chickens havinghigh maternal antibody levels

Saitoh et al. inserted cDNA encoding F and HN proteins of NDV into theHVT genome (WO 99/18215). The inserted genes were under the control ofthe CMV or RSV promoter. The foreign gene insertion site was a newlyidentified intergenic region between UL44 and 45 or between UL45 and 46.These recombinants conferred good protection against NDV challenge inSPF chickens as well as in chickens with NDV maternal antibodies.However, these recombinants expressed two inserted genes of HN and F.Since HN protein induces heamagulutination inhibition (HI) antibodies toimmunized chickens, it is difficult to distinguish vaccine immunizedchickens and NDV infected chickens. Therefore, a recombinant virusexpressing F protein gene, which induces an adequate protective immunityagainst Newcastle disease, is a more desirable vaccine. This objectivewas not easily attainable because rHVT having only F gene of NDV didn'tinduce desirable immunity in chickens as indicated by Morgan et al.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of activities of deleted beta-actin promoter;

FIG. 2 shows a schematic diagram of construction of intermediate plasmidpGIPecF;

FIG. 3 shows a schematic diagram of construction of homology plasmidp45/46PecF;

FIG. 4 shows a Western blot assay;

FIG. 5 shows an ELISA titer of survived and non-survived SPF chickens;and

FIG. 6 shows an ELISA titer of survived and non-survived commercialbirds.

SUMMARY OF THE INVENTION

The present invention provides a recombinant herpesvirus of turkeysmodified by the insertion of cDNA of the F protein of NDV under thecontrol of a modified chicken beta-action promoter (Pec promoter). TherHVT induces long lasting protective immunity against NDV in SPFchickens as well as in commercial chickens that have high maternalantibody to NDV.

Specifically, the present invention provides a recombinant herpesvirusof turkeys modified by the insertion of the NDV F gene under the controlof a promoter, of which sequence is described in SEQ NO.1. The presentinvention further provides a Newcastle Disease-Marek's Disease bivalentvaccine consisting mainly of the said recombinant herpesvirus ofturkeys.

The present invention is described below in more detail.

SEQ ID NO.1 5′-AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATTGGCCCCGCCGGCTGACCGCCCACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCATTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGATGCAGTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGGCGGGGCGGGGCGAGGGGGGCGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGAGTGACCGCGTCTAGAGG-3′(NDV F gene)

As long as encoding the NDV F protein, any gene from any NDV strain isappropriate for the purpose of the present invention. The F gene ofSato, Miyadera, D26, Atami, or Fuji as well as of Texas GB, B1, orLaSota strain is an example. The F gene of a field isolate or of anyknown DNA sequence is also appropriate. Among these, the gene from D26is a favorable example. Incorporating an additional antigen gene intothe backbone virus is not desirable for the purpose of the presentinvention.

(Promoter)

In the present invention, the NDV F gene is controlled by a promoterdescribed in SEQ NO.1 (designated Pec promoter) or that homologous toit. The Pec promoter (Japanese Unexamined Patent Publication No.2001-188) is generated by deleting a dispensable region of the chickenbeta-actin promoter. A promoter homologous to Pec means a promoter ofwhich activity is nearly equal to that of Pec and of which length is 120to 850 base pair (bp) or more favorably 150 to 600 bp. A homologouspromoter can be generated by substitution, deletion, or addition ofnucleotides of to the beta-actin promoter. The promoter used in thepresent invention may include a naturally occurring or modified enhancersequence. An example of such a promoter is COA promoter described in SEQNO.2.

(Avian Herpesvirus)

As long as being non-pathogenic to chickens, any herpesvirus of turkeyscan be used in the present invention. For instances, FC126 (ATCCVR-584B), PB-THV1, H-2, YT-7, WTHV-1, or HPRS-26 strain is suitable forthe backbone virus. Among these, FC126 is favorably used in the presentinvention because of its safe use in chickens.

(Region for Gene Insertion)

Several non-essential regions of HVT are known, which are dispensablefor virus growth and suitable for NDV F gene insertion. For instance,UL43 (WO 89/01040), US2 (WO 93/25665) or inter-ORF region between UL44and UL46 (WO 99/18215) is appropriate for the insertion of the F gene.Among these, the inter-ORF region between UL44 and UL46 is mostsuitable. For the present invention, a non-essential region can newly beidentified by the following general procedure. First, avian herpesvirusDNA fragments of appropriate length are cloned into an E. coli plasmidand physically mapped by restriction enzyme analysis. Then a genecassette consisting of a promoter and a marker gene is inserted into anappropriate restriction site of the cloned DNA fragment resulting in ahomology plasmid. As described later, if the homologous recombinationwith the obtained homology plasmid resulted in a recombinant virusexpressing the inserted marker gene and if it were stable in vitro andin vivo, the originally selected DNA fragment should be a non-essentialregion suitable for NDV-F cDNA insertion.

(Construction of rHVT)

For the present invention, any known method of generating therecombinant avian herpesvirus is applicable. A typical example is asfollows. (1) First, as described above, a recombinant plasmid isconstructed, which includes a non-essential region of the avianherpesvirus. Then, preferably with a promoter at the 5′ terminus and apolyadenlyation signal at the 3′ terminus, NDV-F cDNA is inserted intothe said non-essential region to generate a homology plasmid. (2) Theresultant plasmid is transfected into chicken embryo fibroblast (CEF)cells infected with parent HVT or co-transfected into CEF cells withinfectious HVT genomic DNA. Transfection is performed by any knownmethod (3) The transfected CEF cells are inoculated into culture platesand incubated till the virus plaques become visible. (4) Theidentifiable plaques include recombinant viruses as well as parentwild-type viruses. The recombinant virus is purified from wild typevirus by any known method to screen expression of inserted foreigngenes.

(Newcastle Disease-Marek's Disease Bivalent Vaccine)

Since the F protein is a protective antigen of NDV and the backbone HVTis a live Marek's disease vaccine, rHVT containing the F gene of thepresent invention may be used bivalent vaccine against Newcastle andMarek's diseases or as monovalent vaccine against Newcastle disease.

The vaccine consisting mainly of rHVT of the present invention mayinclude chicken cells and/or ingredients of culture media. As long asnot pharmacologically detrimental, the vaccine may contain anyingredients such as preservatives. In addition, the vaccine of thepresent invention can be used as a mixture with any recombinant ornon-recombinant viruses such as the MDV serotype 1 or serotype 2 vaccinestrains.

Any known method is applicable to the preparation of the recombinantbivalent vaccine of the present invention. For instance, rHVT isinoculated into permissive culture cells such as CEF cells and grown toan appropriate titer. Then, the cells are scraped off from cultureplates or bottles by scraper or by trypsin treatment and subjected tocentrifugation. Cells separated from the supernatant are then suspendedin the culture medium containing dimethyl sulfoxide and stored in liquidnitrogen. When rHVTs are in the supernatant, they are collected andlyophilized.

The bivalent recombinant HVT vaccine is administered to chickens by anyknown method of inoculating Marek's disease vaccine. For instance, thevaccine of the present invention is diluted to give 10-10⁵, or morefavorably 10²-10⁴ plaque forming units (PFU)/dose, and inoculated intosubcutaneously behind the neck of one day of age chickens or intoembryonating eggs by syringe or by any apparatus for injection.

The present avian bivalent vaccine gives SPF chickens 90% or moreprotection against the NDV challenge and at least 70% or more protectionto the commercial chickens having a significant level of anti-NDVmaternal antibody.

In the present invention, protection against NDV challenge is determinedby the ratio of protected birds to total tested birds in the challengetesting as described in the examples. First, the appropriate dose of theNDV challenge virus is determined by challenging non-vaccinated birds.90% or more of these birds (the negative control group) must showclinical signs. Next, the vaccinated birds are challenged with the samedose of the challenge virus by intra-muscular route to the femoralregion, or by intra-tracheal, intra-ocular, or infraorbital sinus route.The challenged birds were observed for onset of Newcastle disease,specificly neurological symptoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

Construction of the Homology Vector

The plasmid construction was essentially performed by the standardmolecular biology techniques (Molecular Cloning: A Laboratory Manual.2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.1989). DNA restriction fragments were electrophoresed on agarose gelsand purified with QIAquick Gel Extraction Kit (QIAGEN, Cat # 28704).

1-1 Construction of the Plasmid Used for the Measurement of the PromoterActivity

Plasmid pGIMICSpolyASfi (2773 bp, WO 99/18215) was generated byintroducing a polyadenylation signal and a SfiI site into themulti-cloning site of pUC18. pGIMICSpolyASfi was then digested with NheIand ligated with the 1721 bp fragment which was excised from Pica geneEnhancer2 (5064 bp, TOYO INK MFG. CO., LTD.) by NheI and XbaI digestion.The obtained plasmid was designated pLUC-Pro (4494 bp).

Next, a DNA fragment (about 1.5 kilobases) including the beta-actinpromoter was prepared by PCR using a chicken cell genomic DNA bank as atemplate. The primers were PrBac1 (SEQ ID NO. 2,5′-CAGTGTCGCTGCAGCTCAGTGCATGCACGCTCATTGCCC-3′) and PrBac2 (SEQ ID NO. 3,5′-GCTCTAGAGTCGACAAGCTTCATGGCTGGCTGCGGAGGAACAGAGAAGGG-3′).

The obtained fragment was digested with PstI and XbaI and ligated withthe 4482 bp fragment generated by digesting pLUC-Pro with PstI and XbaI.The resultant plasmid, designated pLUC-bac, was 5986 bp long andincluded the beta-actin promoter region.

pLUC-bac was digested with BamHI and BglII and the resulting 5958 bpfragment was self-ligated to yield pLUC-bac-Sma. Since pLUC-bac-Sma hadPstI and SmaI sites upstream of the beta-actin promoter, restriction ofpLUC-bac-Sma with PstI and SmaI followed by self-ligation yielded thefirst plasmid to obtain the shortened 5′-region of the beta-actinpromoter. Similarly, restriction of pLUC-bac with SacI and XbaI followedby self-ligation generated the second plasmid to obtain the shorted3′-region of the beta-actin promoter. The resultant two plasmids werethen linearized and subjected to the ExoIII treatment usingKilo-Sequence Deletion Kit (TAKARA SYUZO CO., LTD.). Sampling wasperformed at one, two, and three minutes to obtain 7 samples from thefirst plasmid and 22 from the second. All 29 fragments included about200 to 1500 bp promoter region. The obtained fragments were blunt-endedand self-ligated by the standard procedure to generate the circularplasmids.

The measurement of the luciferase activity was performed with PicaGene(TOYO INK MFG. CO., LTD.). Briefly, one μg each of 29 plasmids wasintroduced into CEF cells by electroporation using Gene Pulser (Bio-RadLaboratories). Cells were then cultured for 24 hours, and cell lysisbuffer (included in PicaGene kit) was added. The cells were frozen forone hour at −80° C., and thawed at room temperature to comp ete the celllysis. After centrifugation, 10 μl of the recovered supernatant wasadded to 100 μl of the PicaGene substrate solution and one minute later,intensity of luminescence (Unit: RLU) was measured with Lumino- Meter(Model 11253, Bio-Rad Laboratories). The Lumino-Meter was used tocalculate the amount of the produced luciferase, an indicator of thepromoter activity (see FIG. 1).

As shown in FIG. 1, among the samples having a deletion towards5′-terminus, those lacking more than 120 bp upstream of the TATAAA boxshowed a drastic decrease in promoter activity. On the contrary, therewas no co-relation between the promoter activity and the length of thedeletion towards the 3′-terminus. For instance, a deletion of about 100bp incurred about a one-fifth decrease in the promoter activity whencompared with that of the full length beta-actin promoter. Furtherdeletion (greater than 100 bp) towards the 3′-terminus did not regainthe original activity.

1-2 Core Sequence Promoter

References from the literature (Ref. 12-19) indicate all regions of thebeta-action promoter are not indispensable. Based on this information,273, 211, 175, and 163 bp fragments were obtained by PCR using thebeta-actin promoter of pLUC-bac as a template. Primers were SEQ NO. 4(5′-TATTTTGTGCAGCGAT-3′) and SEQ NO. 5(5′-ACGTCTAGAAGGCAACGCAGCGACT-3′), or SEQ NO. 4 and SEQ NO. 6,(5′-CTGTCTAGATAACGCGGTCAGTCAGA-3′). The obtained fragments coa273,coa211, coa175 and coa163 were called Core Sequence Promoters (COAs).

Next, these four COAs were digested with PstI and XbaI and resultingfragments were ligated with a fragment of 4482 bp, which was excisedfrom pLUC-pro with PstI and XbaI. The promoter activity of these fourplasmids was measured as described in Example 1-1. In consequence, theactivity of pULC-COA273, pLUC-COA211, pULC-COA175 and pLUC-COA163 was97%, 76%, 96%, and 34%, respectively, of that of the full-lengthbeta-actin promoter. The promoter included in pULC-COA273 was designatedthe COA Promoter, since it showed the highest promoter activity.

1-3 Construction of p45/46Sfi (FIGS. 2 & 3)

Based on the information of the gC homologue (gCh) gene of MDV serotype1 (Coussens et al., J. Virol. 62:2373-2379, 1988) and its adjacentBamHI-B fragment (Japanese Unexamined Patent Publication No. H6-292583),a DNA fragment having an SfiI site between two ORFs UL45h and UL46h, wasprepared by PCR and cloned into pUC18. First, HVT DNA was prepared fromCEF cells infected with the HVT FC126 strain according to the method ofLee et al. (J. Gen. Virol., 51: 245-253, 1980). Using the obtained HVTDNA as a template, PCR was performed with two pairs of primers. Thefirst pair was SEQ NO. 7 (5′-CCCCGAATTCATGGAAGAAATTTCC-3′) and SEQ NO. 8(5′-CGCGGGCCTTATTGGCCAAAACACACCTCTAACGGTTACT-3′). The second pair wasSEQ NO. 9 (5′-GCGCGGCCAATAAGGCCAAAACACAGTAACCGTTAGAGGT-3′) and SEQ NO.10 (5′-CCCCAAGCTTTCAAGTGATACTGCGTGA-3′). Using the mixture of theobtained two PCR products as template, another PCR was conducted withSEQ NO. 7and SEQ NO. 8 to generate a fragment having an SfiI sitebetween two ORFs UL45h and UL46h.

The resulting fragment was then digested with EcoRI and HindIII andligated to pUC18, which had been digested with EcoRI and HindIII. Theobtained plasmid was designated p45/46Sfi.

1-4 Construction of pUC18polyASfi

Plasmid pUC18polyASfi was constructed by annealing two syntheticoligonucleotides (SEQ NO. 11 and SEQ NO. 12) followed by ligation topUC18, which had been digested with EcoRI and HindIII.

SEQ NO. 11 138bp: 5′-AGCTTGCCAATAAGGCTGCAGGTCGACTCTAGAGGATCCCCGGGCGAGCTCGCTAGCGGGCCCGCATGCGGTACCGTCGACAATAAAGAACCGCTTTAAGAATAGTGTTTATTTTTGTGTTTATGGCCAATAAGGCCG-3′ SEQ NO. 12 138bp:5′-AATTCGGCCTTATTGGCCATAAACACAAAAATAAACACTATTCTTAAAGCGGTTCTTTATTGTCGACGGTACCGCATGCGGGCCCGCTAGCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCTTATTGGC-3′

The inserted fragment was excised by BglI and the cohesive ends weredesigned to be inserted into the SfiI site of p45/46Sfi. In addition, amulti-cloning site was added to the 5′-terminus of the fragment so thata promoter-foreign gene cassette could be inserted. The 43 bp sequencedownstream from the SalI site was polyadenlyation signal, which wasderived from the sequence located downstream of UL46h of the MDV GAstrain.

1-5 Construction of pBac

An DNA fragment of about 1.5 kilobases (kb) including the beta-actinpromoter described in EXAMPLE 1-1 was digested with PstI and SalI andligated with pUC18polyASfi, which had been digested with PstI and SalI.The obtained plasmid was designated pBac.

1-6 Construction of pGICOA

Using pBac as a template, PCR was performed with primers PrBac3 andPrBac4.

(SEQ NO. 13) PrBac3: 5′-TTTCTGCAGTATTTTGTGCAGCGAT-3′ (SEQ NO. 14)PrBac4: 5′-CTGTCTAGATAACGCGGTCAGTCAGA-3′

PrBac3 has a PstI site and PrBac4 has an XbaI site. The PCR-amplifiedfragment was excised with PstI and XbaI to generate a fragment of about300 bp. The fragment was then ligated to pUC18polyASfi, which had beendigested with PstI and XbaI. The obtained plasmid was designated pGICOA.

1-7 Construction of Pec Promoter

The CMV enhancer was added to the COA promoter to enhance its promoteractivity.

Since the CMV enhancer had two BglI sites, a BglI cassette from the CMVenhancer was not easily inserted into the SfiI site of pGICOA. To deleteBglI sites, in vitro mutagenesis was conducted by PCR with three pairsof primers using pBK-CMV (Stratagene) as a template. The primers werePrCMV1 (SEQ NO. 15) and PrCMV3 (SEQ NO. 17), PrCMV4 (SEQ NO. 18) andPrCMV5 (SEQ NO. 19), and PrCMV6 (SEQ NO. 20) and PrCMV2 (SEQ NO. 16).Using the obtained three amplified fragments as a template, thesecondary PCR was performed with primers PrCMV1 and PrCMV2. Since PrCMV1had a PstI site and PrCMV2 had an EcoT22I site, the amplified fragmentwas digested with PstI and EcoT22I to yield a fragment of about 300 bp,which was in turn cloned into the PstI site of pGICOA to generatepGIPec. The promoter included in pGIPec, designated Pec promoter,consisted of about 275 bp fragment from the CMV enhancer followed by a273 bp fragment from the beta-actin promoter. The Pec promoter showedenhanced promoter activity, 6.5 times higher than that of COA promoterwhen evaluated in vitro as described in Example 1-1.

(SEQ No. 15) PrCMV1: 5′-GGG CTG CAG AGT TAT TAA TAG TAA TCA ATT-3′ (SEQNo. 16) PrCMV2: 5′-GGG ATG CAT CCA TTT ACC GTC ATT GAC GTC-3′ (SEQ No.17) PrCMV3: 5′-GGG TCG TTG GGC GGT CAG CCG GCG G-3′ (SEQ No. 18) PrCMV4:5′-CTT ACG GTA AAT GGC CCG CCC GCT G-3′ (SEQ No. 19) PrCMV5: 5′-TAC ACTTGA TGT ACT GCC AAT GGG C-3′ (SEQ No. 20) PrCMV6: 5′-TAT TTA CGG TAA ACTCCC CAT TGG C-3′1-8 Construction of NDV-F Vector

Using XLIII10H (Sato, H. et al., Virus Research, 7241-7255, 1987) as atemplate, PCR was performed with primers PrF1 and PrF2.

(SEQ No. 21) PrF1: 5′-GCTCTAGAGGATCCGCATGGGCTCCAGATCTTCTACCAGGATCCC-3′(SEQ No. 22) PrF2: 5′-GCGAGCTCGGTCCATGACTGAAGACTGCTATTGG-3′

PrF1 has XbaI and BamHI sites. PrF2 has a SacI site.

The amplified fragment of about 1.9 kb long encoded 553 amino acids ofthe F gene, which was identical to that reported in Virus Research,7241-7255, 1987. The fragment was digested with XbaI and KpnI and clonedinto pGIPec, which had been digested with XbaI and KpnI. The obtainedplasmid was designated pGIPecF.

1-9 Construction of the Homology Vector p45/46PecF

For construction of the homology plasmid, p45/46pecF, the Pec promoter,the NDV F gene and the SV40 polyadenylation signal sequence was insertedin p45/46Sfi. First, the Pec promoter and the NDV F gene were excisedfrom pGIPecF with BglI and KpnI. Second, the SV40 polyadenylation signalsequence was amplified from pBR-CMV (Stratagene) by PCR and cut withBglI and KpnI. These two fragments were cloned into the SfiI sitedisrupting one of the SfiI sites and resulting in the homology plasmid,p45/46pecF.

EXAMPLE 2 Construction and Purification of rHVT/NDV

Viral DNA of the HVT wild type, FC126 strain (wt-HVT) was prepared asdescribed by Morgan et al. (Avian Diseases, 34:345-351, 1990). The CEFcells were transfected with the prepared wt-HVT DNA and p45/46PecF (seeExample 3-3). The resulting recombinant virus was plaque purified bystaining plaques with the anti-NDV-F antibody.

Briefly, 5 μg of the homology vector p45/46PecF and 25 μg of wt-HVT DNAwere dissolved in 100 μl of Saline G (0.14 M NaCl, 0.5 mM KCl, 1.1 mMNa₂HPO₄, 1.5 mM NaH₂PO4, 0.5 mM MgCl₂, 0.011% glucose). Next, CEF cellswere suspended in 0.7 ml of Saline G and subjected to electroporation.Transfected cells were incubated for 10 minutes at room temperature andtransferred to a 60-mm dish, which contained 5 ml medium consisting ofLeibovitz's L-15 (GIBCO BRL, Cat. #41300-39) and McCoy's 5A Medium(GIBCO BRL, Cat. #21500-061) (1:1) and 4% calf serum (LM (+) medium).After incubating at 37° C. in 5% CO₂, recombinants were purified fromwt-HVT by a series of limiting dilutions. Expression of the F gene wasconfirmed at each round of purification using an antigen-antibodyreaction using the anti-NDV-F monoclonal antibody 3-1G/5 (Morrison, T.G., Proc. Natl. Acad. Sci. U.S.A. 84: 1020-1024, 1987) as the primaryantibody. The purification procedure was repeated until every obtainedplaque was stained positively by the anti-NDV-F antibody. The purifiedrecombinant HVT was designated rHVT/NDV.

EXAMPLE 3

Verification of the Stability of rHVT/NDV

3-1 Southern Hybridization

The purified rHVT/NDV was propagated on CEF cells of two 150-mm dishesto obtain confluent plaques. Cells were recovered from dishes byscraping, transferred to Falcon tubes and centrifuged at 1,500revolutions per minute (rpm) for 5 minutes. Harvested cells were washedwith phosphate buffered saline (PBS), re-suspended in 1.2 ml of PBS and0.8 ml of a lysis buffer (1.25% TritonX-100, 250 mM 2-ME, and 50 mM EDTAin PBS) and lysed by vortexing for 30 seconds. The lysates were thencentrifuged at 3,000 rpm for 5 minutes at room temperature and thesupernatant was transferred to an Eppendorf tube. The viruses werecollected by centrifugation at 15,000 rpm, 22° C. for 20 minutes. Therecovered pellets were then suspended in 1 ml of 12.5 mM Tris-Cl (pH7.5) supplemented with 4 μl of a nuclease solution (0.25 mg/ml DNase 1,0.25 mg/ml RNase A, 150 mM NaCl), incubated at 37° C. for 30 minutes,and disrupted by incubating at 55° C. for 30 minutes with a SDS-proteasesolution (500 mM EDTA, 25 μl; 10% SDS, 125 μl; H₂O, 87 μl; 10 mg/mlProtease K, 12.5 μl; 2-Mercaptoethanol, 0.5 μl). The obtained mixturewas treated twice with phenol-chloroform, and 16 μl of 5M NaCl was addedto the aqueous phase. The viral DNA was precipitated by adding 2.5volumes of −20° C. ethanol, washed with 70% ethanol and centrifuged.After air-drying, the recovered pellets were dissolved in 50 μl of TEbuffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA).

The recovered viral DNA in TE buffer was then digested with XhoI, XbaI,and SfuI and subjected to 0.8% agarose gel electrophoresis. Theelectrophoresed DNA fragments on the single gel were transferredsimultaneously to two nylon membranes (Molecular Cloning: A LaboratoryManual, third edition, 6.35, Sambrook, J., and Russell, D. W. ColdSpring Harbor Laboratory). After fixing DNA by baking, the immobilizedDNA was hybridized with a DIG-labeled probe, “F probe” or “IS45/46probe”, which was prepared with PCR DIG Probe Synthesis Kit (ROCHEDIAGNOSTICS, Cat. #1636090). The F probe was prepared by PCR withNDV-F-F (SEQ ID NO. 23) and NDV-F-R (SEQ NO. 24) as primers andp45/46PecF as a template. IS45/46 probe was prepared with 45/46-F (SEQNO. 25) and 45/46-R (SEQ NO. 26) and p45/46Sfi.

NDV-F-F □SEQ NO. 23□ 5′-CTAGCAGTGGCAGTTGGGAAGAT-3′ NDV-F-R □SEQ NO. 24□5′-GTTAAGGCAGGGGAAGTGATTTGT-3′ 45/46-F □SEQ NO. 25□5′-GGGGAAGTCTTCCGGTTAAGGGAC-3′ 45/46-R □SEQ NO. 26□5′-GGTGCAATTCGTAAGACCGATGGG-3′

The results of southern blotting showed that a 3.6 kb fragmenthybridized to the F probe and 3.6 and 1.2 kb fragments hybridized to theIS45/46 probe, indicating that the obtained rHVT/NDV had the expectedgenomic structure.

3-2 In vitro Stability of rHVT/NDV

rHVT/NDV was passaged ten times in CEF cells and subjected to Southernblot analysis. The results were the same with those obtained in Example3-1, indicating that rHVT/NDV of the present invention was stable evenafter the 10 passages.

3-3 In vivo Stability of rHVT/NDV

3000 PFU of rHVT/NDV was inoculated subcutaneously into the back of theSPF chicken or of commercial chicken having the anti-NDV maternalantibody. At week 3, 4, 5, and 6 post vaccination, peripheral blood wascollected from the vaccinated birds and viruses were recovered fromlymphocytes in the peripheral blood. Harvesting the lymphocytes wasperformed as follows.

Using a 2.5 ml syringe containing 0.5 ml of a heparin solution (100u/ml), 2 ml of blood was collected from chickens and placed over 5 ml ofFicoll-Paque (Amersham-Pharmacia) in a 15 ml Falcon centrifuge tube.After centrifugation at 1800 rpm for 20 minutes, a band of peripheralblood lymphocytes was formed at the boundary between the Ficoll-Paqueand serum layers. The boundary layer was collected with a Pasteurpipette, inoculated into CEF cells in a 9 cm dish and cultivated forseven days. Isolation of viruses from lymphocytes was consideredsuccessful when plaques were observed during the process of the firstcultivation followed by the subcultivation. The results were summarizedin Table 1.

TABLE 1 Isolation of rHVT from peripheral blood lymphocytes ofvaccinated birds rHVT/NDV HVT FC126 Non-vaccinated MA+*** SPF**** MA+SPF MA+ 3 wks** 2/2* ND***** 2/2 1/1 ND 4 wks 3/3 2/2 1/3 2/2 0/2 5 wks3/3 2/2 1/3 ND 0/2 6 wks 3/3 2/2 1/3 2/2 0/2 *Number of birds from whichthe virus was recovered / Number of tested birds **Weeks of age***Maternal antibody positive chicken ****Specific pathogen free chicken*****Not done

As shown in Table 1, rHVT/NDV as well as the parent HVT FC126 strain wasrecovered from peripheral blood lymphocytes of vaccinated chickens fromthree to six weeks post inoculation. When stained with the anti-Fantibody as described in Example 2, all recombinant plaques were shownto express the F gene, indicating that rHVT/NDV was stable after in vivopassage.

EXAMPLE 4

Verification of the Inserted Gene Expression by rHVT/NDV

4-1 Immunofluorescence Technique

rHVT/NDV-infected cells were incubated with fresh CEF cells in tissueculture chamber slides at 37° C. and obtained plaques were fixed withcold acetone. To detect the expressed antigen gene products, the chickenanti-NDV antiserum or rabbit anti-F antiserum was used in the 500-folddilution in PBS as the primary antibody. Fixed cells were incubated withthe primary antibody at room temperature in 100% humidity for about onehour, washed three times with PBS, and reacted with the secondaryantibody for about one hour at room temperature. The anti-chickenimmunoglobulin or anti-rabbit IgG antibody, which was labeled with afluorescent substance (FITC), was used as the secondary antibody. Afterwashing three times with PBS, the treated slides were inspected byfluorescence microscopy. Cells infected with parent HVT FC-126 were usedas a control. The results were summarized in Table 2.

TABLE 2 Expression of the inserted F gene by rHVT/NDV (Detection offluorescence) Primary antibody anti-VP2 rabbit anti-F chicken anti-NDVmonoclonal Virus antiserum antiserum antibody □R63□ PBS rHVT/NDV + + − −FC126 − − − − None − − − − +: detected, −: not detectedAs Shown in Table 2, rHVT/NDV Expressed the Inserted NDV-F Gene.4-2 Western Blotting

CEF cells were infected with rHVT/NDV at m.o.i.=0.1, incubated for 72hours, and solubilized in a SDS-GEL loading buffer. Similarly, cellsinfected with parent HVT FC126 or non-infected cells were incubated andsolubilized. The obtained samples were reduced, denatured, and subjectedto SDS-PAGE. The electrophoresed proteins were transferred from SDS-GELto a PVDF membrane (Immobilon-P, Millipore), which was blocked in 1% w/vnon-fat milk powder in PBS at room temperature for one hour. The treatedmembrane was then reacted with the anti-F rabbit antiserum in 500-folddilution at room temperature, washed three times with PBS, and incubatedfor one hour with the biotinylated anti-rabbit goat antiserum. Afterwashing three times with PBS, the membrane was incubated for one hourwith an avidin-alkaline phosphatase complex, washed three times with PBSand one time with TBS, and reacted with BCIP-NBT (a substrate ofalkaline phosphatase.) As shown in FIG. 4, a protein band of 60kilodaltons (kDa) was observed only in the lane with rHVT/NDV infectedcells, which was the expected size of the F protein.

EXAMPLE 5

Efficacy of rHVT/NDV in SPF Chickens

rHVT/NDV obtained in Example 2 was subjected to the efficacy test as aNewcastle disease vaccine.

1,950 PFU/100 μl/bird of rHVT/NDV were inoculated subcutaneously intothe back of fifteen one-day-old SPF chickens (LineM, Japan BiologicalLaboratories) using 20 Gauge syringe. From three weeks post vaccinationonward, the serum was collected from the vaccinated birds and anti-NDVantibody titer was measured by a commercial ELISA kit (IDEXX, ELISA kitto diagnose Newcastle Disease). Chickens of the positive control groupwere vaccinated at 14 day of age with a commercial NDV live vaccineaccording to the vender's recommendation. Chickens of the negativecontrol group were not administered with any vaccine. At 43 days of age(42 days post vaccination), chickens of all three groups were challengedwith 10³EID₅₀ of NDV-TexasGB, the standard challenge strain in theUnited States, by intra-muscular route to the femoral region. Thechallenged chickens were inspected daily to check mortality or to detectany onset of Newcastle disease.

TABLE 3 Challenge experiments of rHVT/NDV-vaccinated SPF chickens withvirulent NDV Dose No. of (PFU/ No. of symptom/total HI (ELISA) titerELISA titer at Vaccination chicken) chickens (%) at hatch challengerHVT/NDV 1950 20  0/20 (0) 0 0.207 ± 0.03 Commercial NDV On label 10 0/10 (0) 1.089 ± 0.29 Live vaccine Positive Controls N/A 10 11/12 (92)0.089 ± 0.01 Negative Controls N/A 5  0/5 (0) N/A

As shown in Table 3, chickens vaccinated with rHVT/NDV did not show anyclinical signs and the ELISA titer at the day of challenge wassignificantly elevated.

EXAMPLE 6

Efficacy of rHVT/NDV in NDV Maternal Antibody Positive Chickens

To examine the efficacy of rHVT/NDV as a vaccine with anti-NDV maternalantibody positive chickens, fertilized eggs of commercial chickens(Hy-line, Kanagawa Youkei Rengoukai) were purchased and incubated. 1,950PFU/μl of rHVT/NDV were inoculated into 18-day-old embryos using a 20Gauge-1.5 inch syringe. Chickens of the positive control group werevaccinated at 14 days of age with a commercial NDV live vaccineaccording to the vender's recommendation. Chickens of the negativecontrol group were not administered with any vaccine. At 43 days of age,chickens of all groups were challenged with 10³EID₅₀ of NDV-TexasGB, thestandard challenge strain in the United States, by intra-muscular routeto the femoral region. The challenged birds were inspected daily tocheck the mortality or to detect any onset of Newcastle disease.

TABLE 4 Challenge experiments of rHVT-vaccinated commercial chickenswith virulent NDV Dose No. of (PFU/ No. of symptom/total HI (ELISA)titer ELISA titer at Vaccination chicken) chickens (%) at hatchchallenge rHVT/NDV 1950 10  0/12 (0) N/A 0.471 ± 0.108 Commercial NDV Onlabel 10  0/10 (0) N/A 1.386 ± 0.287 Live vaccine Positive Controls N/A10 10/10 (100) N/A 0.047 ± 0.003 Negative Controls N/A 5  0/5 (0) N/AN/A As shown in Table 4, chickens vaccinated with rHVT did not show anyclinical sign and ELISA titer at the day of challenge was significantlyelevated.

EXAMPLE 7

Co-Relation Between the ELISA Titer and Vaccine Efficacy

Co-relation between the ELISA titer and protection was assessed bymeasuring the anti-NDV titer in the sera collected at the day ofchallenge from chickens, which were vaccinated as described in Example 5and 6. The antibody titer was measured by the commercial ELISA kitdescribed in Example 5. FIGS. 5 and 6 show the ELISA titers of survivedor non-survived SPF and commercial birds, respectively. As indicated,all chickens having 0.15 or more of the antibody titer (S/P value)survived the virulent NDV challenge.

EXAMPLE 8

Duration of Immunity

Five chickens vaccinated with rHVT/NDV as described in Example 6 werekept without challenge and every two weeks the NDV-ELISA titers of thecollected sera were measured. As a control, non-vaccinated chickens weresubjected to the same procedure. The S/P value 0.15, obtained in Example7, was used as a criterion to determine the protection. The results areshown in Table 5.

TABLE 5 Duration of protective immunity conferred by rHVT/NDV Weeks ofage Group 2 3 4 5 7 11 15 20 24 30 45 50 rHVT/NDV antibody titer 0.850.50 0.31 0.21 0.36 0.84 0.91 0.88 1.21 0.90 1.04 1.12 protection YesYes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Non-vaccinated antibodytiter 0.74 0.46 0.10 0.03 −0.01   0.02 0.05 0.02 0.02 0.00 0.01 0.02Controls protection Yes Yes No No No No No No No No No No

As shown in Table 5, at 4 weeks of age, the ELISA titer of thenon-vaccinated chickens decreased to the value lower than the criticalone, indicating no protection. By 50 weeks of age, the titer decreasedto nearly zero. On the contrary, the vaccinated chickens showed thelowest titer at 5 weeks of age, which was high enough to confer thecomplete protection. Afterwards, the value continued to increasegradually to 1.21 at 24 weeks of age, and remained the same until 50weeks of age. These data indicate that the rHVT/NDV of the presentinvention is capable of inducing long-lasting protective immunity in thevaccinated maternal antibody-positive commercial birds.

EXAMPLE 9

Isolation of rHVT/NDV from Peripheral Blood Lymphocytes of rHVT/NDVVaccinated Chickens

3000 PFU of rHVT/NDV was inoculated subcutaneously into the back of aday old anti-NDV maternal antibody positive commercial chickens. Thechickens were kept for 30 weeks and every two weeks, peripheral bloodwas collected from the vein of the wing web of the vaccinated birds.Viruses were recovered from lymphocytes in peripheral blood as describedin Example 3. Viruses were recovered from all chickens vaccinated withrHVT/NDV and all viruses were shown to express the F gene.

1. A recombinant herpesvirus of turkeys harboring an F protein gene ofNewcastle disease virus under the control of a promoter of whichsequence is shown in SEQ No.1; wherein said recombinant herpesvirus ofturkeys does not comprise an HN gene.
 2. A recombinant herpesvirus ofturkeys as in claim 1 wherein the promoter and F protein gene areinserted into a noncoding, inter-ORF region of the backbone virusgenome.
 3. A recombinant herpesvirus of turkeys as in claim 2 whereinthe said noncoding region is that located between UL45 and UL46 of theherpesvirus genome.
 4. A method of inducing protective immunity in anavian host against avian herpesvirus and Newcastle disease virus, whichmethod comprises inoculating the avian host with the recombinantherpesvirus of turkeys as in claim 1, 2 or
 3. 5. A method of inducingprotective immunity in an avian host as in claim 4 wherein a recombinantherpesvirus of turkeys is administered to the avian host by subcutaneousor in ovo route.
 6. A poultry vaccine comprising a recombinantherpesvirus of turkeys as in claim 1, 2, or 3.